US5587291A - Method for the determination of plasmin α2 -antiplasmin complexes and the use of this method as a mean of determining changes in the fibrinolytic system - Google Patents

Method for the determination of plasmin α2 -antiplasmin complexes and the use of this method as a mean of determining changes in the fibrinolytic system Download PDF

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US5587291A
US5587291A US08/157,210 US15721093A US5587291A US 5587291 A US5587291 A US 5587291A US 15721093 A US15721093 A US 15721093A US 5587291 A US5587291 A US 5587291A
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pap
complex
plasmin
serum
plasma
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Bernd Binder
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Berbi GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/968—Plasmin, i.e. fibrinolysin

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  • the invention describes a method for the quantitative determination of plasmin- ⁇ 2-antiplasmin complexes as well as the use of this method as a mean of determining changes in the fibrinolytic system.
  • ⁇ 2-antiplasmin is the most important plasmin inhibitor. Its rapid reaction with plasmin results in the formation of an inactive complex composed of one molecule of each component. Two steps are involved in this process: first, a reversible complex is formed between the lysine-binding site of plasmin and complementary sites on the carboxy terminal end of the ⁇ 2-antiplasmin molecule. In a second step, an irreversible complex is generated associated with the cleavage of a peptide-bond in the inhibitor. During activation of plasminogen to plasmin an equilibrium exists between formation of plasmin- ⁇ 2-antiplasmin complexes, occurring preferentially in the fluid phase and binding and action of plasmin on the fibrin surface. Bound to fibrin, plasmin is protected against inhibition by ⁇ 2-antiplasmin. However, whenever fibrin is completely dissolved the plasmin liberated from the fibrin surface is immediately complexed by ⁇ 2-antiplasmin.
  • the amount of PAP complexes generated in serum can be expected to be dependent on the amount of other plasminogen activators present in the respective environement as well as on plasminogen activator inhibitors, receptors and binding proteins.
  • thrombolytic therapy leads to extensive activation of the fibrinolytic system to a massive plasmin formation in the fluid phase and maximal PAP complex formation.
  • nonfibrin specific plasminogen activators such as streptokinase or urokinase can cause complete consumption of plasmin inhibitors resulting in an increased bleeding tendency due to plasminemia. Therefore the plasmin activity is no longer restricted to its specific substrate fibrin but can extend to nonspecific substrates as fibrinogen and other coagulation factors.
  • plasmin- ⁇ 2-antiplasmin complexes can serve as indicative for general plasminemia during hyperfibrinolytic states with fibrinogen and ⁇ 2-antiplasmin consumption and possible bleeding tendency; on the other hand, slightly increased levels of plasmin- ⁇ 2-antiplasmin complexes are indicative for ongoing thrombus formation and thrombus dissolution as in the case of thrombophilia and PAP complexes in serum, respectively and are dependent on the total fibrinolytic potential.
  • Harpel et al. first published a sensitive assay with a polyclonal antibody against ⁇ 2-antiplasmin as catching antibody and POX-labeled Fab-fragments against plasminogen as detecting system, whereby the Fab- fragments are preferred to whole antibodies to reduce unspecific binding of plasminogen and other plasma proteins to immuno-globulins.
  • Other ELISA-systems were described by Holvoet et al. and Mimuro et al.
  • the invention here is a test system employing a specific monoclonal antibody against the neoantigen in the PAP complex.
  • Said antibody was obtained by immunizing with a natural not ammonia-cleaved plasmin-anti-plasmin complex and said antibody furthermore reacts also exclusively with a plasmin-anti-plasmin complex utilizing Western Blot and said antibody is utilized as a catching antibody.
  • the respective plasmin-anti-plasmin complex ELISA is also highly specific and sensitive in plasma and serum samples.
  • the antibody utilized in the invention MPW7AP is secreted from the respective hybridoma cell line and the said was deposited at the Public Health Laboratory Service, European Collection of Animal Cell Cultures, Salisbury, UK, with the Number 94072843.
  • the hybridoma cell line was obtained according to published methods by fusion of spleen cells of a mouse immunized with the native plasmin-anti-plasmin complexe with a mouse myeloma cell line (NS0).
  • the secreted monoclonal antibody is characterized by a specific reaction exclusively with plasmin-anti-plasmin complexes but not with the respective separate components utilizing Western Blot technique (FIG. 1).
  • the test described here is a solid phase enzyme immunoassay in which MPW7AP, a specific monoclonal antibody directed against the neoantigen of the PAP-complex is adsorbed on plastic microtiter plates. During incubation with test samples PAP-complexes are selectively bound and after washing away unbound material the complexes are detected by MPW2PG POX, a peroxidase-labelled monoclonal antibody against the kringle 1-3 region of the plasminogen part of the complex. Quantification of labelled antigen-antibody conjugates is achieved by ABTS, a chromogenic substrate for peroxidase.
  • 96-well flat bottom microtiter plates with high binding capacity e.g. NUNC immunoplate maxisorp 4-93454 or GREINER ELISA plates (No. 655061), plate sealers (e.g. COSTAR 3095).
  • ELISA reader for 405 nm and 492 nm wavelength (e.g. Anthos reader 2001, Zinsser Austria)
  • serum albumin bovine purified (BSA) ORHO 20/21, Behring, Germany
  • Coating buffer 1.59 g Na2CO3.10H2O, 2.93 g NaHCO3 100 mg THIMEROSAL® with distilled water to 11, pH 9.6.
  • Coating solution 20 ⁇ g/ml MPW7AP in coating buffer; 100 ⁇ l/well
  • PBS Phosphate buffered saline
  • Washing buffer PBS+0.5% Tween 20
  • Dilution buffer 1 PBS+1% BSA+2000 KIU/ml aprotinin+20 mM benzamidinium chloride or a specific inhibitor for t-PA, e.g. PPACK
  • Dilution buffer 2 (DB2): DB1+1% PAP depleted plasma
  • Dilution buffer 3 (DB3): DB1+10% PAP depleted plasma
  • Substrate buffer 1.29 g citric acid monohydrate, 1.375 g Na2HPO4.2H2O with distilled water to 100 ml, pH 4.0
  • Substrate solution 1 mg ABTS/ml solution+1 ⁇ l H2O2 30%/ml solution in substrate buffer; 100 ⁇ l/well
  • Coating buffer and coated plates containing an antimicrobial substance are stable at 4° C.
  • Other protein containing solutions and washing buffer should be prepared freshly or kept under sterile conditions to prevent microbial growth.
  • Antibiotics should not be used in these cases to avoid adverse reactions with the peroxidase.
  • the wells of an ELISA plate are filled with 100 ⁇ l/well coating solution, preferably by use of a multichannel micropipette.
  • the plate should remain covered with a self-adhesive plastic foil for at least 16 h at 4° C. but can also be stored that way for prolonged time.
  • the plate is emptied and refilled with 100 ⁇ l/well of blocking solution and incubated for 1 h at 37° C. to block excessive reactive groups on the plate surface. For all incubation steps the plate remains covered with the plastic foil.
  • Normal citrated or EDTA plasma can be used but one should be aware that uninhibited plasminogen activators will lead to in vitro formation of PAP complexes.
  • blood samples should therefore always be collected using anticoagulants containing inhibitors e.g. 2000 KIU/ml aprotinin+20 mM benzamidine final concentration.
  • the plasma samples are diluted 1:10 in DB1 for low concentrations and 1:100 for high concentrations of PAP complexes. 100 ⁇ l/well are needed and at least duplicate determinations are recommended.
  • PAP complex standard plasma is reconstituted with distilled water. Serial dilutions from 1:100 to 1:800 and blanks are prepared in DB3 for use with low PAP concentration samples and in DB2 for use with samples containing high PAP concentrations. Also 100 ⁇ l/well and duplicates are necessary.
  • the plates are washed three times with approximately 300 ⁇ l/well washing buffer either manually or by use of an automatic plate washer. After each emptying step the plate is carefully tapped dry on absorbent paper towels.
  • the reading of the samples are compared with those of a reference PAP complex preparation.
  • plasmin- ⁇ 2-antiplasmin complexes are not easily prepared in a stable purified form--the complex is susceptible to proteolytic degradation and different epitopes might be generated whether the complex is formed in excess of plasmin or of inhibitor (1)--we decided to prepare PAP complexes directly in plasma and to calibrate them with PAP complexes generated from purified components. For this purpose citrated plasma was incubated with more than saturating concentrations of urokinase to produce maximum amount of plasmin- ⁇ 2-antiplasmin complexes. Thereafter the reaction was terminated by addition of benzamidine and TRASYLOL®. The so formed complexes are stable either frozen at -70° C. or lyophilized at 4° C.
  • plasmin was prepared freshly from purified Glu-plasminogen by incubation with urokinase bound to SEPHAROSETM(beaded agarose) and after removal of urokinase SEPHAROSETM the resulting plasmin activity was determined with S-2251 immediately and after 30 min incubation with purified ⁇ 2-antiplasmin at 37° C. The differences in plasmin activity represents the amount of PAP complexes formed (FIG. 2).
  • the described test is specific for plasmin- ⁇ 2-antiplasmin complexes and allows full plasma recovery provided the standard was diluted in PAP- depleted plasma in the same concentration as the plasma samples to be tested.
  • the lower detection limit is 10 ng/ml in purified systems as well as in plasma.
  • PAP values in citrated plasma are 152 ⁇ 72 ng/ml and 132 ⁇ 72 ng/ml in EDTA plasma; in serum samples PAP values are mostly increased.
  • Thrombolytic therapy results in PAP plasma values of >800 ng/ml.
  • PAP values were found to be increased 12 months thereafter (FIG. 3).
  • FIG. 1 is Western Blot analysis of 3 monoclonal antibodies using ⁇ 2-antiplasmin, one of the components of the plasmin-anti-plasmin complex as antigen.
  • Antibody 3 is Antibody MPW7AP
  • FIG. 2 is a graph which compares PAP complexes prepared in plasma with PAP complexes from purified components. In the insert the amidolytic determination of plasmin activity from freshly prepared plasmin incubated with buffer or without excess ⁇ 2-antiplasmin is shown. From the differences in plasmin activity (plasmin inhibited) the concentration of found complexes is calculated.
  • FIG. 3 is a graph which compares PAP-complexes in controls and in patients with coronary heart disease.
  • FIG. 4 Time is a graph which plots the time of plasmin-anti-plasmin complex formation against concentration in the serum of a reactive patient undergoing therapy with aspirin
  • FIG. 5 is a set of four graphs which plot the time of generation of plasmin-anti-plasmin complexes in serum (0-4 Hours) before ( ⁇ ) one week ( ⁇ ) two weeks ( ⁇ ) after initiation of Aspirin treatment and one week after cessation of Aspirin treatment ( ⁇ ).

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US08/157,210 1992-04-14 1993-04-14 Method for the determination of plasmin α2 -antiplasmin complexes and the use of this method as a mean of determining changes in the fibrinolytic system Expired - Lifetime US5587291A (en)

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AT0077292A AT398004B (de) 1992-04-14 1992-04-14 Verfahren zur bestimmung von plasmin-alpha2- antiplasmin-komplexen unter verwendung eines komplexspezifischen monoklonalen antikörpers
AT772/92 1992-04-14
PCT/AT1993/000065 WO1993021532A1 (de) 1992-04-14 1993-04-14 METHODE ZUR BESTIMMUNG VON PLASMIN-α2-ANTIPLASMIN KOMPLEXEN SOWIE VERWENDUNG DER BESTIMMUNGSMETHODE ALS MASS FÜR VERÄNDERUNGEN IM FIBRINOLYTISCHEN SYSTEM

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5888749A (en) * 1990-12-20 1999-03-30 Iatron Laboratories, Inc. Anti-human plasmin-α2 -plasmin inhibitor complex antibodies, hybridomas, and immunological determination method
US20020192794A1 (en) * 1999-11-13 2002-12-19 Dadd Christopher A. Process for the production of a reversibly inactive acidified plasmin composition
US20090275513A1 (en) * 1999-11-13 2009-11-05 Rebbeor James F Composition and method for preparing plasminogen
US20110201077A1 (en) * 2008-06-04 2011-08-18 Talecris Biotherapeutics ,Inc. Composition, method, and kit for preparing plasmin
US9206410B2 (en) 2009-03-03 2015-12-08 Grifols Therapeutics Inc. Compositions, methods and kits for preparing plasminogen and plasmin prepared therefrom
US10670611B2 (en) 2014-09-26 2020-06-02 Somalogic, Inc. Cardiovascular risk event prediction and uses thereof
CN114736948A (zh) * 2022-06-10 2022-07-12 深圳市帝迈生物技术有限公司 一种α2-抗纤溶酶活性测定试剂盒

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5888749A (en) * 1990-12-20 1999-03-30 Iatron Laboratories, Inc. Anti-human plasmin-α2 -plasmin inhibitor complex antibodies, hybridomas, and immunological determination method
US9879246B2 (en) 1999-11-13 2018-01-30 Grifols Therapeutics Inc. Reversibly inactivated acidified plasmin composition
US20020192794A1 (en) * 1999-11-13 2002-12-19 Dadd Christopher A. Process for the production of a reversibly inactive acidified plasmin composition
US20030012778A1 (en) * 1999-11-13 2003-01-16 Zimmerman Thomas P. Reversibly inactivated acidified plasmin
US20080311105A1 (en) * 1999-11-13 2008-12-18 Zimmerman Thomas P Reversibly Inactivated Acidified Plasmin
US20090275513A1 (en) * 1999-11-13 2009-11-05 Rebbeor James F Composition and method for preparing plasminogen
US7871608B2 (en) 1999-11-13 2011-01-18 Talecris Biotherapeutics, Inc. Reversibly inactivated acidified plasmin
US8268782B2 (en) 1999-11-13 2012-09-18 Grifols Therapeutics Inc. Composition and method for preparing plasminogen
US20110201077A1 (en) * 2008-06-04 2011-08-18 Talecris Biotherapeutics ,Inc. Composition, method, and kit for preparing plasmin
US8617863B2 (en) 2008-06-04 2013-12-31 Grifols Therapeutics Inc. Composition, method, and kit for preparing plasmin
US9206410B2 (en) 2009-03-03 2015-12-08 Grifols Therapeutics Inc. Compositions, methods and kits for preparing plasminogen and plasmin prepared therefrom
US10670611B2 (en) 2014-09-26 2020-06-02 Somalogic, Inc. Cardiovascular risk event prediction and uses thereof
CN114736948A (zh) * 2022-06-10 2022-07-12 深圳市帝迈生物技术有限公司 一种α2-抗纤溶酶活性测定试剂盒

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JPH06508219A (ja) 1994-09-14
ATA77292A (de) 1993-12-15
AU3817693A (en) 1993-11-18
EP0589011A1 (de) 1994-03-30
WO1993021532A1 (de) 1993-10-28
AT398004B (de) 1994-08-25

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